MX2011001637A - COMPOSITIONS OF NISINA LIQUIDA. - Google Patents

Abstract

The present invention relates to liquid nisin compositions that have a high antimicrobial activity. The invention further relates to a method for preparing liquid nisin compositions as well as their use as a preservative in food products.

Description

COMPOSITIONS OF LIQUID NISINE FIELD OF THE INVENTION The present invention relates to liquid nisin compositions, to methods for preparing the compositions, to their use as a preservative and to methods for preserving the foods in which the compositions are used.

BACKGROUND OF THE INVENTION The need for improved methods for food preservation is great. It has been estimated that a large part of the global food supply is lost as a result of microbial spoilage and microbial infections transmitted by food pose a constant and serious threat to human health.

Several bacterial species that can contaminate and grow in food and crops are pathogenic or produce toxins and generate a range of food poisoning diseases. Despite the substantial improvement in technology and hygiene, food products can be exposed to deterioration and pathogenic bacteria in the environment in which food is handled and the number of food poisoning is still increasing in most countries. Food preservation techniques, for example heat processing, freezing, ultrasound, irradiation, and high-pressure treatment, significantly reduce the antimicrobial load, but the evidence that processed foods are contaminated with microorganisms after processing is of particular interest. and before packing. Microbial problems related to various foods such as dairy and meat products, fresh and chilled foods and seafood are of increasing concern in the food industry.

Especially food products in the pH range of 4.5 to 7.0 are known to be susceptible to microbial spoilage by microorganisms, which include pathogens and spore-forming bacteria. At lower pH levels, yeasts, molds and acid tolerant bacteria are more relevant. Above all, processed foods are not consumed directly after processing, thus allowing bacteria to survive the production process or to be introduced and grown by post-contamination. Because food consumption can occur without the reheating of processed foods at sufficient temperatures and for sufficient time, there is a risk of food poisoning or food spoilage.

Additionally, the recent trend of minimally processed foods with the intrinsic sensory and nutritional qualities of raw materials and fresh foods has raised the risk. Moderate conservation treatments, such as high hydrostatic pressure and pulsed electric field techniques have proven successful, but often rely on effective barriers, ie cold chain and the addition of natural antimicrobials.

A thorough investigation has been made in the field of food safety to develop designs of effective antimicrobial products, resulting in a combination of compositions, processing and preservation.

Nisin is an antibacterial substance similar to peptide produced by Lactococcus lactis subsp. lactis It comprises 34 amino acids and is active against mainly gram-positive bacteria. Nisin is non-toxic and free of side effects. Nisin is generally recognized as a safe substance and is widely used in a variety of foods. Examples of such products are processed cheese, milk, curdled cream, dairy desserts, ice cream mixes, liquid egg, baked flour products, dressings and beer. Nisin is stable to heat and survives at pasteurization temperatures with minimal loss of activity.

Usually, nisin is obtained by fermenting a species of Lactococcus lactis and is further formulated as a dry powder that can be used as a preservative as such or after it has first been resolved in a suitable solvent. Delvoplus® and Nisaplin® are commercial names for nisin powder containing 1 million IU per gram. They are distributed by DSM and Danisco, respectively. These nisin powder products have several disadvantages: dust is generated after handling, and it is difficult to dose and mix small amounts of powder in the products. Therefore, liquid nisin compositions that do not have the disadvantages described above are commercially preferred.

Liquid nisin compositions as such are known in the art. Although it has been reported that the liquid nisin compositions have activity against gram-positive bacteria (see Mota-Meira et al (2000), Montville et al (1999), US 5,584,199 and US 4,597,972) and even gram-negative bacteria ( see EP 0 453 860, US 5,260,271 and US 5,559,096), there are still liquid nisin compositions having improved antimicrobial activity, particularly against gram-positive bacteria found in the food industry.

SUMMARY OF THE INVENTION Surprisingly, nisin compositions having a very high activity against gram-positive bacteria have now been found. Due to their high antimicrobial activity, only low amounts of the compositions are required for effective action against bacteria, for example gram-positive bacteria. The compositions have good microbiological stability which in combination with their good physical and chemical stability makes the compositions suitable for prolonged storage and therefore gives them a long shelf life. Additionally, the compositions of the invention may have a low turbidity, which makes them suitable for use in food applications, where the addition of low turbidity additives is of importance. In clarity of the above characteristics, the compositions of. The invention can be used advantageously as food preservatives.

DETAILED DESCRIPTION OF THE INVENTION According to a first aspect the invention provides a method for preparing a liquid nisin composition, preferably an aqueous liquid nisin composition. The method comprises the steps of: a) preparing a first liquid nisin containing the composition having a pH of from about 1.5 to about 12, preferably about 3 to about 10, preferably about 3.5 to about 9.5, more preferably about 4 to about 9 , still more preferably about 4.5 to about 8.5, still more preferably about 5 to about 8, more preferably about 5.5 to about 7.5, and in particular about 5.5 below 7, b) isolating the solid compounds from the first prepared liquid nisin which contains the composition, c) contacting the isolated solid compounds with a solution having a pH of from about 0 to about 5, preferably about 0.5 to about 4.5, more preferably about 1 to about 4, still more preferably about 1.5 to about about 3.5, more preferably about 1.5 to about 3 and in particular about 2 to about 3 to prepare a second liquid nisin composition, and d) to remove the solid compounds from the second liquid nisin composition. Step d is optional, but in a preferred embodiment it is developed in the method of the invention.

In a further embodiment the method of the invention comprises the step of: e) adjusting the pH of the second liquid nisin composition to a desired pH value such as a pH between 2 and 6, for example a pH between 2 and 3 or at pH between 5 and 6.

Optionally, at least one of the additional functional compounds mentioned below may be added before, during or after at least one of the steps of the method of the invention. For example, a cryoprotectant, for example glycerol, can be added during step c, such that the second liquid nisin composition comprises 35% to 60% w / w of cryoprotectant. In another example, a compound that reduces or decreases foaming and / or an additional antimicrobial compound, for example an organic acid or a salt thereof, can be added before step b. In a preferred embodiment, however, at least one additional functional compound is added after step d and before step e, or during or after step e.

In one step of the embodiment comprises mixing nisin with an aqueous solution to prepare a first liquid nisin containing the composition having a final inorganic salt (for example NaCl) in concentration of 1.5 or less, preferably 0.05 M to 1.5 M and more preferably 0.1 to 1.5 M. The first liquid nisin containing the composition has a pH of from about 1.5 to about 12, preferably about 3 to about 10, preferably about 3.5 to about 9.5, more preferably about 4 to about 9, even more preferably about 4.5 to about 8.5, still still more preferably about 5 to about 8 and more preferably about 5.5 to about 7.5 and in particular about 5.5 to below 7. Any nisin source can be suspended and / or dissolved in the aqueous solution. In a preferred embodiment the nisin is a powder, preferably a dry powder. For example, commercially available nisin powder compositions such as Delvoplus® and Nisaplin® can be used. The source may comprise nisin A, nisin Z or a combination thereof. The aqueous solution may be a buffer solution, for example a phosphate buffer such as NaH2P04 / Na2HP0. Other suitable shock absorbers can also be used frequently. These include, but are not limited to, acetate buffers, lactate buffers, citrate buffers, glycine / HCl buffers and any combination thereof.

The solid compounds can be separated / isolated from the first liquid nisin containing the composition by well-known isolation techniques. In a preferred embodiment step b is developed by means of centrifugation, filtration or any combination thereof.

Subsequently, a second liquid nisin composition can be prepared by for example contacting, for example dissolving or mixing or suspending, the isolated solid compounds with / in a solution, preferably an aqueous solution, having a pH of from about 0 to about 5, preferably about 0.5 to about 4.5, more preferably about 1 to about 4, even more preferably about 1.5 to about 3.5, more preferably about 1.5 to about 3 and in particular about 2 to about 3. In one embodiment an additional functional compound mentioned Forward is added during this stage.

Then, the second liquid nisin composition can be purified by removing for example the remaining residues and / or proteins without nisin or parts thereof. This purification step can be developed by well-known isolation techniques. In a preferred embodiment step d is developed by means of centrifugation, filtration or any combination thereof.

The method described above results in a liquid nisin composition having a much higher activity against microorganisms, particularly gram-positive bacteria, than the liquid nisin compositions described in the prior art. In other words, the method of the present invention results in liquid nisin compositions having a much lower inhibitory concentration (MIC) against microorganisms, particularly gram-positive bacteria, than the liquid nisin compositions described in the prior art.

Therefore, a nisin composition obtainable by a method according to the invention is another part of the present invention. The nisin composition can be solid, but preferably this is a liquid composition.

In one embodiment the nisin compositions of the invention have a MIC of 1.0 g / mL or less against at least one gram-positive bacteria. The MIC refers to the minimum concentration of a compound or composition necessary to inhibit the growth of the tested organism. Preferably, the MIC is an average of at least three independent repeats. The compositions of the present invention have a MIC of 1.0 pg / mL or less when tested for the growth of the inhibition of at least one gram-positive bacteria in the assay described above. In one embodiment the compositions of the invention have a MIC of 0.5 pg / mL or less, preferably a MIC of 0.1 and g / mL or less, more preferably a MIC of 0.05 pg / mL or less, even more preferably a MIC of 0.01 pg. / mL or less, still more preferably a MIC of 0.005 pg / mL or less, particularly a MIC of 0.001 pg / mL or less, more particularly a MIC of 0.0005 pg / mL or less against at least one gram-positive bacteria and more particularly a MIC of 0.0001 pg / mL or less against at least one gram-positive bacteria. Gram-positive bacteria include, but are not limited to, Micrococcus sp. , Listeria sp. , Bacillus sp. , Staphylococcus sp. , Clostridium sp. ,, Streptococcus sp. , Lactobacillus sp. and Lactococcus sp. In one embodiment the gram-positive bacterium is selected from the group consisting of Bacillus, Lactococcus, Staphylococcus, Listeria and Micrococcus. Suitable species within the genus Bacillus, Lactococcus, Staphylococcus, Listeria and Micrococcus include, but are not limited to, B. subtilis, L. lactis, S. aureus, L. innocua and M. luteus, respectively. Within species that give suitable strains include, but are not limited to, Bacillus subtilis ATCC 31578, Lactococcus lactis ATCC 19257, Staphylococcus aureus ATCC 27661, Listeria innocua LMD 92.20 and Micrococcus luteus B212, respectively.

In a preferred embodiment the compositions of the present invention have a MIC of 0.5 and g / mL or less, preferably a MIC of 0.1 and g / mL or less, more preferably a MIC of 0.05 and g / mL or less, even more preferably a MIC of 0.01 and g / mL or less, still more preferably a MIC of 0.005 and g / mL or less, particularly a MIC of 0.001 and g / mL or less and more paricularly a MIC of 0.0005 and g / mL or less against at least one strain of luteus, preferably M. luteus B212.

The nisin activity can be measured using the following bioassay well known to the skilled person (see Pongtharangkul and Demirci, 2004), which includes pretreating the nisin composition at low pH. In summary, M. luteus B212 containing agar plates (Iso-sensitest agar) is prepared using a culture that has grown recently. After drying, a vacuum pump is used to create small holes in the agar. The samples and their dilutions (10 μ?) Are transferred to the holes and allowed to diffuse into the agar for 18 hours at 5 ° C. Subsequently, the agar plates are incubated for 24 hours at 30 ° C and the zones of inhibition are measured around the sample containing the holes. Parallel to the samples, controls with known amounts of nisin (0 - 1600 IU / mL) are included. Their zones of inhibition are used to prepare a calibration curve required to determine the nisin levels of the samples. All stages are carried out aseptically. The UI for Nisin has already been defined as follows. The World Health Organization Committee on Biological Standardization, twenty-second report. Technical Report of the World Health Organization series, No. 444 in 1970, has established an international reference nisin preparation, and the international unit (hereafter IU) is defined as 0.001 mg of this preparation. Delvoplus® and Nisaplin®, trade names for nisin powder products that contain 1 million IU per gram, are distributed by DSM and Danisco, respectively. By means of the above test the nisin concentration in the samples can be determined.

The nisin MIC compositions can be measured by the following MIC assay. The nisin activity is measured using the standard microdilution broth assay well known to the skilled person. In summary, a Micrococcus luteus B212 containing the Iso-sensitest culture broth is prepared using a culture that has grown recently. The number of cells per mL is determined using a counting chamber. Preferably, a cell count of 103 is used. 100 L of inoculum is added to each well of a 96-well microtiter plate. 100 of a nisin composition is added to the first well (Al) and mixed properly by pipetting up and down three times. A serial dilution is made by transferring 100 μL from the first well to the next well (A2) and diluting appropriately. This is repeated until each component is diluted serially in 36 wells. Then, the plates are incubated at 30 ° C for 7 days and read each day for bacterial growth. MIC concentrations are the lowest concentration that completely inhibits growth.

In one embodiment the compositions of the invention have a pH of about 0 to about 5, preferably about 0.5 to about 4.5, more preferably about 1 to about 4, even more preferably about 1.5 to about 3.5, more preferably about 1.5 to about 3 and in particular about 2 to about 3. In such pH conditions, the microbiological stability of the compositions of the invention is good and the MIC of the compositions is low and stable during storage.

In a further embodiment the compositions according to the invention comprise 0.01 to 5%, preferably 0.05 to 2.5%, more preferably 0.1 to 1.0%, more preferably 0.15 to 0.5% and in particular 0.2 to 0.3% (w / w) of nisin .

The nisin compositions of the invention may comprise a low amount of salts such as inorganic salts for example NaCl. It is understood that it does not mean that the additional functional compounds mentioned below (for example antimicrobial compounds such as organic acids or their salts) are included within the definition of "salt". In one embodiment the compositions of the invention comprise a salt, for example inorganic salt, for the nisin ratio of 100: 1 to 1: 100, preferably 50: 1 to 1: 100, more preferably 25: 1 to 1: 100 and in particular 10: 1 to 1: 100. In one embodiment the nisin compositions of the invention are essentially free of salts, preferably inorganic salts such as for example NaCl. The inorganic salt can be any suitable, food grade inorganic salt. Examples of inorganic salts are NaCl, Na2SO4, (Ca) 3 (P04) 2, K 03, KC1 and MgC03. The concentration of these salts in the compositions is 100 mg / mL or less, preferably 50 mg / mL or less, more preferably 25 mg / mL or less and in particular 15 mg / mL or less. The salt concentration can be measured by separate cation analysis, by atomic absorption anion analysis, by HPLC or preferably by determination of the ash content by ignition (550 +/- 25 ° C). The nisin compositions having a low concentration of inorganic salts are very attractive, because they will not interfere with the food matrix to give unwanted reactions and alterations of taste and / or structure.

The nisin compositions of the invention may comprise low amounts of different nisin and salt components. These components can be proteins or their parts. It is understood that it does not mean that the additional functional compounds mentioned below [for example antimicrobial compounds, antifoaming agents, surfactants, etc.) are included within the definition of "components other than nisin and salt". In one embodiment the compositions of the invention comprise a different nisin component for the nisin ratio of 100: 1 to 1: 100, preferably 10: 1 to 1: 100 and more preferably 2: 1 to 1: 100. In one embodiment the nisin compositions of the invention are essentially free of these components. The components can originate from the biomass produced during the nisin fermentation process using Lactococcus lactis. The nisin concentration can be measured first by the assay described above. Subsequently, the total protein concentration can be estimated using classical assays known to the skilled person. The concentration of protein without nisin can be estimated by subtracting the nisin concentration from the total protein concentration.

In yet another embodiment the compositions of the invention. they are clear liquid compositions. The clear compositions of liquid nisin can be used in and / or in any type of product. In view of their clarity, they can be used advantageously in products where clarity is of importance such as gelatin-based products eg desserts, fruit juices, beverages and surface applications on food products. The clear liquid compositions as used herein are the liquid compositions having a turbidity of 0 to 100 FNU, preferably 0 to 50 FNU, more preferably 0 to 25 FNU and particularly 0 to 10 FNU. Turbid liquid compositions are liquid compositions having a turbidity above 100 FNU. The turbidity in FNU (Nephelometric Formazine Unit) can be determined with a light scattering method and can be measured using a Nephla turbidity photometer with the DIN method measured in EN 27027 / ISO 7027. The clear compositions as well as the cloudy compositions of Liquid nisin can be prepared by the method according to the invention. A clear liquid composition is prepared, if a liquid nisin containing the composition having a pH of about 5 or more, preferably a pH of about 5 to about 9, is prepared in step a of the method of the invention. A cloudy liquid composition is prepared, if a liquid nisin containing the composition having a pH below about 5, preferably a pH of about 1.5 below about 5, or a pH above about 9, preferably at a pH above about 9 to about 12 is prepared in step a of the method of the invention. The clear and cloudy compositions of liquid nisin have high activity described above against microorganisms, in particular gram-positive bacteria.

A method- where the final inorganic salt concentration. { for example NaCl) of the first liquid nisin containing the composition (ie the liquid nisin composition prepared in step a of the method according to the invention, see above) that is above 1.5 M has several disadvantages compared to a method wherein the final inorganic salt concentration of the first liquid nisin containing the composition is 1.5 or less. First, the first liquid nisin composition having a final inorganic salt concentration above 1.5 M shows a reduced separation performance in centrifugation (ie has low sedimentation rate) compared to the liquid nisin compositions with a concentration of final inorganic salt of 1.5 M or less. Second, the resulting final liquid nisin composition that is prepared by developing the method according to the present invention (ie, steps a to c and optionally steps d and e, see above), wherein the first liquid nisin containing the composition has a concentration of final inorganic salt above 1.5 M, has several disadvantages: it is cloudy; has a lower purity of the final liquid nisin compositions that have been made by means of a method according to the present invention wherein the first liquid nisin containing the composition contains a final inorganic salt concentration of 1.5 or less; it has a lower antimicrobial activity than that of the final liquid nisin compositions that have been made by means of a method according to the present invention wherein the first liquid nisin containing the composition contains a final inorganic salt concentration of 1.5 or less; Y it has a greater risk of being precipitated than the final liquid nisin compositions which have been made by means of a method according to the present invention wherein the first liquid nisin containing the composition contains a final inorganic salt concentration of 1.5 M or less The liquid nisin compositions of the invention have at least one of the disadvantages mentioned below compared to the liquid nisin preparations known in the art: - the compositions of the invention have a better antimicrobial efficacy compared to liquid nisin compositions of the prior art, and / or the compositions of the invention are essentially free of salts such as for example inorganic salts for example NaCl and essentially free of other components other than nisin. As a result of this, in the food applications, the use of the compositions of the invention that do not interfere with the food matrix to give the undesired taste and / or structure reactions and / or alterations, and / or - the compositions of the invention can be clear, that is they have a low turbidity (between 0 and 100 FNU). Such compositions do not interfere with the color and / or clarity of the products to which they are applied.

- According to another embodiment, the compositions of the invention additionally comprise at least one additional functional compound including, but not limited to, an additional antimicrobial compound such as an acid eg sorbic acid, propionic acid, benzoic acid, acid acetic, lactic acid, citric acid, cinnamic acid, or a salt of any of these acids, a glucose oxidase, natamycin, lysozyme, poly-L-lysine, nystatin, lucensomycin, amphotericin B, philippine, pediocin; a surfactant for example SDS, Tween, fatty acids; an agent that adjusts the pH such as HC1 or NaOH or a buffering agent for example a phosphate salt or acetate salt; a cryoprotectant such as glycerol or propanediol; thickening agents for example xanthan gum, guar gum, arabic gum, tragacanth gum, gellan gum, locust bean gum, carrageenan gum, ramxano gum, alginate, starch, carboxymethylcellulose, carboxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinyl alcohol, polyethylene glycol, polypropylene glycol. Moreover, the compositions of the invention may comprise agents that reduce or decrease foam formation.

Additional compounds can be added to the compositions of the invention in liquid or solid form and can be mixed well in advance or directly before use. Using at least one additional antimicrobial compound / preservative in the nisin compositions of the invention is expected to additionally stabilize it microbiologically and therefore may be beneficial for its shelf life.

The nisin activity present in an aqueous liquid composition can be substantially increased by removing the impurities. Moreover, the solubilization index of nisin in the aqueous compositions is increased by the removal of impurities such as, for example, inorganic salts. Nisin can be partially linked to impurities that result in nisin that is not available for its preservative activity. In other words, nisin has limited bioavailability in the presence of impurities. As used herein, the term "bioavailability" refers to the availability, amount (eg, concentration), or nisin activity in a solid, semi-solid or liquid formulation. Impurities such as proteins without nisin or other nisin components, cell wall debris and salts can have a negative effect on the solubilization index of nisin. It is found that approximately less than 50% of the nisin present in such a liquid formulation is available as a preservative in case these impurities are present. It has been found that impurities are present in commercially available nisin products. Commercially available nisin generally contains 5-25% protein without nisin or cell debris. These impurities originate from the production process of nisin. Recovery, purification or reformulation is frequently used after the fermentation of the salts that are still present in the final nisin formulation.

In a further aspect the invention relates to an aqueous suspension of nisin comprising thickening agents. Of course, two or more different thickening agents can also be used. The suspensions of the invention comprise 0.01 to 5%, preferably 0.05 to 2.5%, more preferably 0.1 to 1%, more preferably 0.15 to 0.5% and in particular 0.2 to 0.3% (w / w) of nisin. The suspensions of the invention comprise 0.01 to 5%, preferably 0.05 to 5%, more preferably 0.1 to 5%, more preferably 0.2 to 5% and in particular 0.5 to 5% (w / w) of thickening agent. The thickening agent is selected from the group consisting of xanthan gum, guar gum, arabic gum, gum tragacanth, gellan gum, locust bean gum, carrageenan gum, ramxano gum, alginate, starch, carboxymethylcellulose, carboxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinyl alcohol , polyethylene glycol and polypropylene glycol. In a preferred embodiment the thickening agent is a gum such as xanthan gum. The pH of the suspension according to the invention is from about 2 to about 12, preferably about 2 to about 11, more preferably about 2 to about 10, even more preferably about 2 to about 9, still even more preferably about 2 to about 8 , more preferably about 2 to about 7 and in particular about 2 to about 6. The suspension of the invention is stable. "Stable suspension" as used herein means a physically stable suspension, i.e. a suspension that shows 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, more preferably 10% or less and in particular 0% sedimentation after storage at room temperature for 9 days at pH 5. The physical stability of the suspensions can be measured by methods known in the art such as the sedimentation test as shown here (see Example 9).

In one embodiment the suspension according to the invention additionally comprises at least one additional functional compound selected from the group consisting of an additional antimicrobial compound, a surfactant, a pH-adjusting agent, and a cryoprotectant. Examples of suitable additional antimicrobial compounds are acids such as sorbic acid, propionic acid, benzoic acid, acetic acid, lactic acid, citric acid, cinnamic acid, acid or salts of any of these acids, a glucose oxidase, natamycin, lysozyme, poly L-lysine, nystatin, lucensomycin, amphotericin B, philippine, pediocin. Examples of suitable surfactants are SDS, Tween, fatty acids, to mention only a few. Examples of suitable pH adjusting agents are among others HC1 or NaOH or buffering agents such as phosphate salts and acetate salts. Examples of suitable cryoprotectants are glycerol and propanediol. Moreover, the suspensions of the invention may comprise agents that reduce or decrease foam formation. Additional compounds can be added to the suspensions of the invention in liquid or solid form and can be mixed either previously or directly before use.

In a further embodiment the invention relates to a method for preparing a suspension according to the invention, the method comprising the steps of: a) adding nisin and thickening agents, separately or as a powder composition, to an aqueous solution. { for example water), and b) mix to obtain a suspension. If necessary, the pH of the suspension can be adjusted to pH from about 2 to about 12, preferably about 2 to about 11, more preferably about 2 to about 10, even more preferably about 2 to about 9, still even more preferably about 2 to about 8, more preferably about 2 to about 7 and in particular about 2 to about 6.

The thickening agent and the nisin can be added separately to the aqueous solution. They can be in powder form or in liquid form. Alternatively, the nisin and the thickening agent may be present in a powder composition and this powder composition may be added to the aqueous solution. In a further embodiment the invention relates to a composition comprising nisin and thickening agents. The nisin and / or thickening agent can be added together with an additional functional compound described above for the aqueous solution and then mixed to obtain a suspension. Alternatively, additional functional compounds can be added after the suspension comprising nisin and the thickening agent has been obtained. In a further embodiment first nisin is added to the aqueous solution, followed by an additional functional compound and then the thickening agent and the mixing solution are added to obtain a suspension. In still a further embodiment, thickening agents are first added to the aqueous solution, followed by an additional functional compound and then nisin is added and the solution is mixed to obtain a suspension. Again in a further embodiment the additional functional compound is first added to the aqueous solution, followed by the addition of thickening agents and / or nisin.

Another aspect of the invention relates to the use of an aqueous suspension according to the invention for the preparation of a treatment liquid for the treatment of a food, concentrate or agricultural product. The liquid treatment can be prepared by mixing an aqueous solution with the suspension according to the invention. The treatment of the food, concentrate or agricultural product can be done by spraying, immersion, immersion, brushing to mention a few.

According to a further aspect, the invention provides the use of a composition or suspension according to the invention as a preservative in and / or on food, concentrate or agricultural product. Hereinafter, the term "suspension" also includes a treatment liquid prepared from a suspension according to the invention. The compositions and suspensions of the invention have no disadvantages associated with powder formulations: they are easier to use (easy dosing) and there is no dusting when used. Additionally, foam formation and dissolution problems that occur when the nisin powder is solubilized in a solvent are avoided. Effective levels of nisin for preserving food products vary from 1 to 1500 IU / g or 0.025 to 37.5 ppm nisin. The compositions and suspensions according to the invention can be used alone, but also in combination with other antimicrobial compositions, for example the compositions comprise organic acids or their salts, lysozyme. The antimicrobial compositions can be applied to the food, concentrate or agricultural product before, during or after the application of the compositions or suspensions according to the invention.

In a further aspect the invention is pertinent to a container comprising 1 to 1000 liter of a composition or suspension according to the invention. The container can be a bottle, bag or tank, to mention only a few i few.

According to a further aspect, the invention provides a method for preserving the food, concentrate or agricultural product, wherein the nisin compositions or suspensions of the invention are used, for example applied in and / or in the respective products. The nisin compositions and suspensions may be applied by spraying, dipping, dipping, brushing, to mention only a few methods. In the case that the substrate / product is a liquid or semiliquid, it can be added directly. The compositions or suspensions may still have a coating, eg, an antimicrobial coating, a substrate a / in which they are applied. Optionally, in an additional step, the product can also be pasteurized / sterilized. This step can of course also be developed before application of the nisin compositions or suspensions of the invention. All types of food products can be treated with the compositions or suspensions of the invention. Food products can be dairy food products; food products that contain or are derived from eggs, meats, especially chicken for example freshly slaughtered chicken, vegetables, crustaceans and fish; rice products such as rice products 3 O boiled; bakery food products; drinks; frozen food products; clear food products such as jelly-based food products such as desserts; juices spreads; jam; canned fruits and other canned products; food products wherein the compositions or suspensions of the invention are applied to the surface. Dairy food products include, but are not limited to, processed milk, milk, cream, milk desserts, ice cream mixes, dressings and yogurts. The compositions and suspensions according to the invention can also be used in the treatment of food packaging and handling equipment and can be included in / on the packaging materials used to package the food, food or agricultural products. The compositions and suspensions of the invention can also be used as a disinfectant to clean surfaces and cook utensils in food processing plants and any area in which foods are prepared or served such as hospitals, clinics, restaurants, especially restaurants of fast food, delicatessen and similar. The compositions and suspensions according to the invention are capable of inhibiting bacterial growth in products over an extended period, for example at least about 1 day, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 days and preferably at least approximately 1000 days. Compositions and suspensions according to the invention can be used to prevent bacterial growth, for example the growth of gram-positive bacteria such as Staphylococcus, Streptococcus, Listeria, and Coryneform bacteria. These can still be used to prevent the growth of gram-negative bacteria such as Gram-negative bacteria such as Salmonella, Shigella, Escherichia Coli, Klebsiella, Pseudomonas, Bacterioides, and Actinobacillus bacteria.

Accordingly, a food, food, or agricultural product comprising a nisin or suspension composition according to the invention is another part of the invention.

In yet another aspect, the invention pertains to a method for producing a solid, for example powder, the nisin composition comprising the step of subjecting the liquid nisin composition according to the invention to, for example, a drying step., lyophilization stage, crystallization stage (if necessary followed by filtration or centrifugation) or a precipitation step (if necessary followed by filtration or centrifugation), to mention only a few. The steps may be developed immediately after step c, d or e of the method for preparing the nisin compositions of the invention as described above. They can also be made after the liquid nisin compositions of the invention have been stored for a period. The resulting solid / powder nisin compositions can be mixed with compositions in. powder comprising other suitable compounds such as for example the additional functional compounds described above.

The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

EXAMPLES Example 1 Preparation of liquid nisin compositions The following liquid nisin compositions are prepared: Composition A: Ten grams of nisin powder - Nisaplin® (Danisco, Denmark) containing 2.5% w / w nisin and at least 50% w / w NaCl is dissolved in an aqueous HC1 solution (pH 2.0-3.0; total volume 100 mi).

Composition B: Ten grams of Nisaplin® nisin powder (Danisco, Denmark) containing 2.5% w / w nisin and at least 50% w / w NaCl is dissolved in an aqueous HC1 solution (H 5.5-6.5; total volume 100 mi).

Compositions C and D: Ten grams of nisin powder Nisaplin® (Danisco, Denmark) containing 2.5% w / w nisin and at least 50% w / w NaCl is dissolved in a buffered aqueous solution of 0.2M dihydrogenphosphate of sodium and disodium hydrogen phosphate (pH 7.0, total volume 100 ml, in addition to the composition having a pH of 6 and making the compositions having a pH of 6.5). The mixture is subsequently mixed for approximately 15 minutes. The mixture is centrifuged at 4,500xg for 15 minutes at 10 ° C and a globule containing nisin is obtained. Subsequently, the globule is dissolved in an aqueous citric acid solution (pH 2.0 to 3.0, total volume 100 ml). The mixture is stirred for 15 minutes. The obtained solution containing nisin is centrifuged at 4,500xg for 15 minutes at 10 ° C to remove the remaining solid components. The liquid compositions obtained are maintained at a pH of 2.0 to 3.0 (Composition C) or the pH is adjusted to a pH between 5.5 and 6.5 by the addition of NaOH (Composition D).

Compositions E and F: The preparation of the compositions E and F is identical to the preparation of the compositions C and D with the proviso that the globule is dissolved in an aqueous HCl solution having a pH of 2.0 to 3.0.

Compositions G and H: Ten grams of Nisaplin® nisin powder (Danisco, Denmark) containing 2.5% w / w nisin and at least 50% w / w NaCl is dissolved in an aqueous HC1 solution (pH 2.0- 3.0, total volume 100 mi). The obtained mixture is dialyzed for 24 hours in an aqueous HC1 solution of pH 2.0 to 3.0. Then, the dialyzed mixture is centrifuged at 4,500xg for 15 minutes at 10 ° C. The liquid compositions obtained are maintained at a pH of 2.0 to 3.0 (composition G) or the pH is adjusted to a pH between 5.5 and 6.5 by the addition of NaOH (Composition H). The compositions obtained are used in the following experiments.

Example 2 MIC assay For the MIC assay, freshly cultured Micrococcus luteus cells (B212) and Pseudomonas aeruginosa cells (ATCC 9027) are obtained from overnight culture growth in Iso Sensitest Culture Broth (Oxoid) and Plaque Counting Culture Broth (Difco) , respectively, at 30 ° C. A stock suspension of 4.3 x 105 and 2.5 x 104 colony forming units CFU / mL, respectively, is prepared in physiological saline. 30 L of the respective stock solution is added to 30 mL of Iso Sensitest Culture Broth (suspension A) and Plate Count Culture Broth (suspension B), respectively. Then, 100 L of suspension A is transferred to each well of a first 96-well microtiter plate and 100 of suspension B is transferred to each well of a second 96-well microtiter plate. The nisin compositions are prepared according to Example 1. 100 and L of a composition are used in a standard microdilution broth assay to determine the Minimum Inhibition Concentration (MIC) of each composition, of nisin. The results, presented in Table 1, show that the nisin compositions C, D, E and F show the highest activity (ie lower MIC) against both microorganisms (a gram-positive microorganism, ie M. luteus, and a gram-negative microorganism, ie P. aeruginosa) at pH 2.5 and pH 6.0. The MIC of compositions C and E is between about 40- to about 100-fold less than the MIC of compositions A and G (all compositions have a pH of 2.5), although the MIC of compositions D and F is significantly lower than the MIC of compositions B and H (all compositions have a pH of 6.0). The MIC of compositions C and D prepared with 0.1 M phosphate buffer at pH 7 is comparable with the MIC of compositions C and D prepared with 0.2 M phosphate buffer at pH 6 or pH 6.5.

In a separate experiment the MIC concentrations of compositions A and C are compared to freshly cultured Bacillus subtilis (ATCC 31578), Staphylococcus aureus (ATCC 27661), Lactococcus lactis (ATCC 19257) and Listeria innocua (LMD92.20). The experiment is made identically to the experiment described above, with the proviso that the prepared stock suspension contains 1.OxlO6, 1.3xl06, 7.7xl04, and 2.8xl05 CFU / mL of the respective microorganism, respectively. The results show that the MIC of the composition C for the gram-positive microorganisms tested is between about 5-about 300-fold lower than the MIC of the composition A (data not shown). The MIC of composition C prepared with 0.1 M phosphate buffer at pH 7 is comparable with the MIC of composition C prepared with 0.2 M phosphate buffer at pH 6 or pH 6.5.

Example 3 Use of liquid nisin compositions in a yeast application Two cakes are prepared. For each cake 1000 grams of Moscovisch Powder Damco are mixed with 800 grams of liquid eggs and 100 grams of water. A cake is prepared by adding 120 mg of Nisaplin® nisin powder (Danisco, Denmark) for liquid eggs. A second cake is prepared by adding 1.2 grams of composition C to liquid eggs. The same amounts of nisin are used in both cakes (150 mg nisin / 1 egg). The mixture is mixed for 10 minutes in a Hobart mixer in the third equipment, and baked in an oven at 170 ° C for 25 minutes. Both cakes are baked in the same way.

The baked cake comprising composition C shows a structure of fine, woolly crumbs, while the one comprising nisin powder shows a structure of irregular, hard crumbs. This clearly shows that the baked goods in which the compositions of the invention are used have a better structure than the baked goods where nisin powder is used. The structure of a baked product is improved by adding a nisin composition according to the invention when compared to adding nisin powder. The example also shows that the compositions according to the invention can be added to the product before baking.

Example 4 Use of liquid nisin compositions in a beverage application In this experiment composition A and C are tested for their ability to reduce the viable count of different contaminating microorganisms in a beverage application. Compositions A and C are prepared according to Example 1, using nisin powder from Silver Elephant, China. The drink used is a malt drink, Bavaria Pony, Colombia. For the experiment, recently cultured Listeria monocytogenes cells (LMD 92.20), Leuconostoc oeno cells (ML-34) and Leuconostoc mesenteroides cells isolated from a contaminated product of a culture grown overnight at 30 ° C in Counting Culture Broth are obtained of Plaques (Difco). The stock suspensions of 3.8xl05, 5.7? 10e and 7.2xl05 CFU / mL, respectively, are prepared in physiological saline solution. 25 μL of the respective stock solution is added to 25 mL of drinking spike with composition A or C. The nisin concentration tested is 0.5 ppm for Listeria monocytogenes cells, 4 ppm for Leuconostoc cells and 2 ppm for Leuconostoc mesenteroides cells. A control that does not comprise nisin is included for each microorganism. The samples are incubated at room temperature and the total count of the microorganisms (in CFU / mL) is measured at different time intervals using well-known methods.

The results are shown in Table 2. They clearly demonstrate that for each of the three different tested microorganisms of composition C it reduces the viable cell count below the limit of detection of 1 CFU / mL in less than a day, while Composition A needs two or three days to accomplish this. Composition C is at least 24-48 hours faster than composition A.

Example 5 Use of liquid nisin compositions in a food application model In this experiment compositions A and C are tested for their ability to reduce the viable count of Listeria monocytogenes in a food application model. After reduction of the viable cell count below the detection limit of 10 CFU / mL the nisin compositions are also tested for their ability to maintain this low level and prevent the spread of the surviving cells. Compositions A and C are prepared according to Example 1, using nisin from Silver Elephant, China. The application model is prepared using a Counting Culture Broth. Plates (Difco). The pH of the semi-solid medium at pH 7.0 with HC1 and autoclaved for 15 minutes at 121 ° C. For the experiment, freshly cultured Listeria monocytogenes cells (LMD 92.20) are obtained from a culture that grows overnight in the Plate Count Culture Broth (Difco) at 30 ° C. A stock suspension of 3.4xl07 CFU / mL is prepared in physiological saline. 250 pL of the stock solution is added to 25 mL of the model medium tipped with composition A or C. The nisin concentration tested is 2.5 and g / mL for the experiment at 10 ° C and 6.25 and 12.5 and g / mL for the experiment at room temperature, a control that does not comprise nisin is included for each temperature tested. The samples are incubated at 10 ° C and at room temperature and the total count of microorganisms (in CFU / mL) is measured at different time intervals using well-known methods.

The results clearly demonstrate that composition C inhibits the extension of hysteria, monocytogenes in a food application model at 10 ° C for more than 25 days, whereas composition A only inhibits extension for four days (see Table 3). The results further show that composition C inhibits the extension of Listeria monocytogenes in a food application model at room temperature for more than 25 days, whereas composition A only inhibits extension for two or three days (see Table 4).

Example 6 Preparation of liquid nisin compositions in pilot scale using centrifugation 100 kg of nisin powder (Silver Elephant, China) is dissolved containing 2.5% w / w nisin and at least 50% w / w NaCl in water. The pH is fixed at 7.0 with NaOH. The mixture is subsequently mixed for about one hour. An antifoaming agent (Clerol FBA 3107) is added to a 1.5 g / kg mixture. The mixture is subjected to continuous centrifugation at 10 ° C to 12,000xg with a feed rate of 200 1 / h. The concentrate containing nisin solids is recovered. Subsequently, the concentrate is dissolved in an aqueous HC1 solution (pH 2.0 to 3.0, mass of complete mixture: 1000 kg). The mixture is stirred for at least one hour. The obtained solution containing nisin is again subjected to continuous centrifugation at 10 ° C to 12,000 xg with a feed rate of 200 1 / h followed by deep filtration (with a filter having a pore size of 3 microns) and filtration Sterile to remove the remaining solid components. The obtained liquid composition is maintained at a pH of 2.0 to 3.0. The turbulence of the final product is 21 FNU, that is, a clear liquid composition is obtained. The liquid nisin composition obtained has a good antimicrobial activity, ie a MIC comparable to the MIC of composition C (see Example 2).

The above process is also developed without developing the second stage of continuous centrifugation. The final product has the same properties as the final product described above.

Additionally, the process is developed without the second stage of continuous centrifugation and without the deep filtration stage. The resulting product also has the same properties as the final product described above.

Example 7 Preparation of liquid nisin compositions on pilot scale using filtration 100 kg of nisin powder (Silver Elephant, China) is dissolved containing 2.5% w / w nisin and at least 50% w / w NaCl in a buffered aqueous solution of 0.2 M sodium dihydrogen phosphate and hydrogen phosphate. disodium (pH 7.0, mass of complete mixture: 1000 kg). The mixture is subsequently mixed for about one hour. The mixture is filtered by filtration with no outlet at 10 ° C using a Dicalite BF filter aid. The filter cake containing nisin solids is recovered. Subsequently, the filter cake is dissolved in a solution of aqueous citric acid (pH 2.0 to 3.0, mass of complete mixture: 1000 kg). The mixture is stirred for at least one hour. The solution obtained containing nisin is subjected to no-leak filtration, deep filtration and sterile filtration at 10 ° C to remove the remaining solid components. The obtained liquid composition is maintained at a pH of 2.0 to 3.0. The turbidity of the final product is 25 FNU, that is, a clear liquid composition is obtained. The liquid nisin composition obtained has a good antimicrobial activity, ie a MIC comparable with the MIC of composition C (see Example 2).

Example 8 Preparation of liquid nisin compositions using low pH filtration 100 g of nisin powder (Silver Elephant, China) is dissolved in water containing 2.5% w / w nisin and at least 50% w / w NaCl. The pH is fixed at 4.0 with NaOH. The mixture is subsequently mixed for about one hour. The mixture is filtered with no output at 10 ° C using filter aid Dicalite BF. The filter cake containing nisin solids is recovered. Subsequently, the filter cake is dissolved in an aqueous HC1 solution (pH 2.0 to 3.0). The mixture is stirred for at least one hour. The obtained nisin-containing solution is filtered using no-exit filtration, deep filtration and sterile filtration at 10 ° C to remove the remaining solid components. The obtained liquid composition is maintained in a 2.0 to 3.0. The turbidity of the final product is 123 FNU, ie a cloudy liquid composition is obtained. The obtained liquid nisin composition has a good antimicrobial activity, ie a MIC comparable with the MIC of composition C (see Example 2).

Example 9 Preparation of stable nisin powder suspension The nisin powder (Silver Elephant, China) containing 2.5% w / w nisin and at least 50% w / w NaCl is suspended in water. Various thickening agents are added in various amounts. The pH is fixed at pH 2 or pH 5 with solutions of HC1 and NaOH. The physical stability of the suspensions is analyzed after storage for 9 days at room temperature by analyzing the height of the frontal sedimentation in a 50 mL tube containing 47.5 mL of the suspension. The results are described in Table 5. The concentration of nisin in all suspensions is 0.25% w / w. Sedimentation is expressed as the percentage of clear liquid that is observed (ie the liquid that does not contain particles). 0% indicates that no sedimentation has occurred and that the suspension therefore has good physical stability. The results show that at pH 2 and pH 5 the suspensions are physically stable when xanthan gum is used at a concentration higher than 0.05% (w / w). The results also show that at pH 2 and pH 5 the suspensions are physically stable when CMC or alginate are used at a concentration of 1% (w / w) or higher, although for HPMC a concentration of 3% (w / w) or higher leads to stable physical nisin suspensions.

Table 1: MIC values of the nisin compositions in yg / mL against M. luteus and P. aeruginosa.

Table 2: Days until the respective microorganism is reduced below the detection limit of 1 CFU / mL in a beverage application with different nisin compositions.

Table 3: Number of days of extension of Listeria monocytogenes is inhibited below the limit of detection of 10 CFU / mL in a food application model at 10 ° C with different nisin compositions.

Table 4: Number of days that the Listeria monocytogenes extension is inhibited below the limit of detection of 10 CFU / mL in a food application model. room temperature with different nisin compositions.

Number of days that extension is inhibited at room temperature Control < l Composition 2 A (250 pg / mL) Composition 3 A (500 pg / mL) Composition > 25 C (250 pg / mL) Composition > 25 C (500 pg / mL) Table 5: Physical stability of the suspension of nisin powder with different thickening agents.

REFERENCES Montville TJ, Chung HJ, Chikindas ML and Chen Y (1999), Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacteriu smegmatis. Letters in Ap l. Microbiol. 28: 189-193. ota-Meira, LaPointe G, Lacroix C and Lavoie MC (2000), MICs of Mutacin B-NY266, Nisin A, Vancomycin, and Oxacillin against bacterial pathogens. Antitnicrobial Agents - and Chemotherapy .44: 24-29.

Pongtharangkul T and Demirci A (2004). Evaluation of agar diffusion bioassay for nisin quantification, Ap l. Microbiol. Biotechnol. 65: 268-272.

Claims (18)

NOVELTY OF THE INVENTION Having described the present invention as above, it is considered as a novelty, and therefore, the content of the following is claimed as property: CLAIMS

1. A method for preparing a liquid nisin composition, comprising the steps of: a) Mixing powdered nisin with an aqueous solution to prepare a first liquid nisin composition having a pH of 3.5 to 12 and a final inorganic salt concentration of 1.5 M or less, b) isolating the solid compounds of the first prepared composition containing liquid nisin by centrifugation, filtration or any combination thereof, c) contacting the isolated solid compounds with a solution having a pH of 1 to 3 to prepare a second liquid nisin composition, and optionally d) removing the solid compounds from the second liquid nisin composition.

2. The method according to claim 1, further comprising the step of: e) adjusting the pH of the second liquid nisin composition to a desired value.

3. A liquid nisin composition obtainable by the method according to claim 1 or 2.

4. The composition according to claim 3 having a minimum inhibitory concentration (MIC) of 0.001 g / mL or less against Micrococcus luteus B212.

5. The composition according to claim 3 or 4 having a pH of 1.5 to 5.

6. The composition according to any one of claims 3 to 5, wherein the composition comprises a salt to nisin ratio of 100: 1 up to 1: 100.

7. The composition according to any one of claims 3 to 6 having a turbidity of 0 to 100 FNU.

8. The composition according to any one of claims 3 to 7, further comprising at least one compound selected from the group consisting of an additional antimicrobial compound, a surfactant, a pH adjusting agent, a cryoprotectant, an antifoaming agent and a thickening agent.

9. An aqueous suspension of nisin having a pH of 2 to 12 and comprising 0.2 to 5% (w / w) of xanthan gum.

10. The suspension according to claim 9, comprising 0.01 to 5% · (w / w) of nisin.

11. The suspension according to any one of claims 9 or 10, further comprising at least one compound selected from the group consisting of an additional antimicrobial compound, a surfactant, an agent 5 that adjusts the pH, an antifoaming agent and a cryoprotectant.

12. A method for preparing a suspension according to any one of claims 9 to 11, the method comprising the steps of: a) add nisin and xanthan gum, either separately or as a powder composition, to an aqueous solution, b) mixing to obtain a suspension comprising 0.2 to 5% (w / w) of xanthan gum, and c) if necessary, adjust the pH of the suspension 5 from 2 to 12. '

13. A powder composition for use in a method according to claim 12 comprising nisin and xanthan gum. '

14. Use of an aqueous suspension as claimed in any one of claims 9 to 11 for the preparation of a treatment liquid for the treatment of a food, concentrate or agricultural product.

15. Use of a composition according to any one of claims 3 to 8 or a suspension according to any one of claims 9 to 11 as a preservative in and / or on a food, concentrate or agricultural product.

16. A method for preserving a food, concentrate or agricultural product, wherein a composition according to any one of claims 3 to 8 or a suspension according to any one of claims 9 to 11 is applied to the food, concentrate or agricultural product .

17. A method for producing a solid nisin composition comprising the step of subjecting a composition according to any one of claims 3 to 8 to a drying step, lyophilization step, crystallization step or precipitation step.

18. a food, concentrate or agricultural product comprising a composition according to any one of claims 3 to 8 or a suspension according to any one of claims 9 to 11.